J Korean Med Sci.  2021 Oct;36(38):e265. 10.3346/jkms.2021.36.e265.

Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells

Affiliations
  • 1Department of Endocrinology and Metabolism, Kyung Hee University Hospital, Seoul, Korea
  • 2Department of Endocrinology and Metabolism, Kyung Hee University School of Medicine, Seoul, Korea
  • 3Department of Biomedical Laboratory Science, College of Health and Medical Sciences, Cheongju University, Cheongju, Korea

Abstract

Background
Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells.
Methods
MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured.
Results
Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone.
Conclusion
Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Keyword

Spironolactone; Osteoblasts; Advanced Glycation End Products; Antioxidants; Mitochondria

Figure

  • Fig. 1 Effects of spironolactone on the MG-induced cytotoxicity in MC3T3-E1 cells. Osteoblasts were treated with SL in the absence or presence of 400 μM of MG for 48 hours, and then (A) cell viability and (B) apoptosis were analyzed. Data are expressed as the mean ± standard error of mean (n = 6).MG = methylglyoxal, SL = spironolactone.#P < 0.05 compared to untreated cells; *P < 0.05 compared to cells treated with MG alone.

  • Fig. 2 Effects of SL on MG-induced osteoblast dysfunction in osteoblastic MC3T3-E1 cells. Cells were treated 10 mM of β-glycerophosphate and 50 μg/mL of ascorbic acid to initiate differentiation. After six days, osteoblasts were treated with SL in the absence or presence of 400 μM of MG for 48 hours, and then (A) collagen content and (B) ALP activity were measured. (C) After 14 days, osteoblasts were treated with SL in the absence or presence of 400 μM of MG for 48 hours, and then mineralization levels were measured. The control values for collagen content, ALP activity, and mineralization were 46.27 ± 0.346 μg/106 cells, 0.502 ± 0.022 unit/mg protein, and 0.654 ± 0.0004 OD/106 cells, respectively. Data are expressed as the mean ± standard error of mean (n = 6).MG = methylglyoxal, SL = spironolactone, ALP = alkaline phosphatase.#P < 0.05 compared to untreated cells; *P < 0.05 compared to cells treated with MG alone.

  • Fig. 3 Effects of SL on ER stress, TNF-α and IL-6 production in MG-treated MC3T3-E1 cells. Osteoblasts were pre-incubated with SL before treatment with 400 μM of MG for 24 hours. The control values for ATF6 and IRE1 were 54.72 ± 0.498 and 3.095 ± 0.179 ng/mg, respectively. The control values for TNF-α and IL-6 were 18.09 ± 2.713 pg/mg and 0.655 ± 0.033 ng/mg, respectively. Data are expressed as the mean ± standard error of mean (n = 6).ATF = activating transcription factor 6, MG = methylglyoxal, SL = spironolactone, IRE1 = inositol-requiring 1, TNF-α = tumor necrosis factor-α, IL = interleukin.#P < 0.05 compared to untreated cells; *P < 0.05 compared to cells treated with MG alone.

  • Fig. 4 Effects of SL on glyoxalase I activity and reduced GSH levels in MG-treated cells. Osteoblasts were pre-incubated with SL before treatment with 400 μM of MG for 48 hours. The control values for (A) glyoxalase I activity and (B) reduced GSH levels were 0.29 ± 0.001 ΔOD/min/mg and 72.03 ± 6.91 μg/mg, respectively. Data are expressed as the mean ± standard error of mean (n = 6).MG = methylglyoxal, SL = spironolactone, GSH = glutathione.#P < 0.05 compared to untreated cells; *P < 0.05 compared to cells treated with MG alone.

  • Fig. 5 Inhibitory effects of SL on MG-induced oxidative stress in MC3T3-E1 cells. Osteoblasts were pre-incubated with SL before treatment with 400 μM of MG for 48 hours. (A) Changes in ROS levels measured using the dichlorodihydrofluorescein fluorescence method. (B) Mitochondrial superoxide levels detected using the MitoSOX™ red mitochondrial superoxide indicator. (C) Cardiolipin oxidation measured using 5 µM NAO. All data are expressed as a percentage of the fluorescence emitted by bound NAO relative to untreated control cells. Note that a decrease in NAO binding is related to cardiolipin peroxidation. Data are expressed as the mean ± standard error of mean (n = 6).ROS = reactive oxygen species, MG = methylglyoxal, SL = spironolactone, NAO = 10-N-nonyl-acridin orange.#P < 0.05 compared to untreated cells; *P < 0.05 compared to cells treated with MG alone.

  • Fig. 6 Effects of SL on MG-induced mitochondrial dysfunction in osteoblastic MC3T3-E1 cells. Osteoblasts were pre-incubated with SL before treatment with 400 μM of MG for 48 hours. (A) Mitochondrial depolarization i.e., loss of MMP is represented by a decrease in the red/green fluorescence ratio; all data are expressed as a percentage of control values. The control values for (B) ATP levels and (C) PGC-1α levels were 1.496 ± 0.193 nmol/mg and 387.98 ± 5.39 ng/mg, respectively. All data for (D) NO are expressed as a mean relative percentage of fluorescence. Data are expressed as the mean ± standard error of mean (n = 6).MMP = mitochondrial membrane potential, MG = methylglyoxal, SL = spironolactone, ATP = adenosine triphosphate, PGC-1α = proliferator-activated receptor gamma coactivator 1α, NO = nitric oxide.#P < 0.05 compared to untreated cells; *P < 0.05 compared to cells treated with MG alone.


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